Chemical formation of hybrid di-nitrogen calls fungal codenitrification into question

Removal of excess nitrogen (N) can best be achieved through denitrification processes that transform N in water and terrestrial ecosystems to di-nitrogen (N₂) gas. The greenhouse gas nitrous oxide (N₂O) is considered an intermediate or end-product in denitrification pathways. Both abiotic and biotic denitrification processes use a single N source to form N₂O. However, N₂ can be formed from two distinct N sources (known as hybrid N₂) through biologically mediated processes of anammox and codenitrification. We questioned if hybrid N₂ produced during fungal incubation at neutral pH could be attributed to abiotic nitrosation and if N₂O was consumed during N₂ formation. Experiments with gas chromatography indicated N₂ was formed in the presence of live and dead fungi and in the absence of fungi, while N₂O steadily increased. We used isotope pairing techniques and confirmed abiotic production of hybrid N₂ under both anoxic and 20% O₂ atmosphere conditions. Our findings question the assumptions that (1) N₂O is an intermediate required for N₂ formation, (2) production of N₂ and N₂O requires anaerobiosis, and (3) hybrid N₂ is evidence of codenitrification and/or anammox. The N cycle framework should include abiotic production of N₂.